JOURNAL OF BACTERIOLOGY, Oct. 1992, p. 6247-6255 Vol. 174, No. 19 0021-9193/92/196247-09$02.00/0 Copyright X 1992, American Society for Microbiology A Chemotactic Signaling Surface on CheY Defined by Suppressors of Flagellar Switch Mutations S. J. ROMAN, M. MEYERS,t K. VOLZ, AND P. MATSUMURA* Department ofMicrobiology and Immunology (MIC 790), University of Illinois, Box 6998, Chicago, Illinois 60680 Received 22 May 1992/Accepted 3 August 1992

CheY is the response regulator that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of has identified fliG and fliM as genes in which mutations occur that allele specifically suppress cheY mutations, indicating interactions among these gene products. We have generated a class of cheY mutations selected for dominant suppression offliG mutations. Interestingly, these cheY mutations dominantly suppressed bothfiG and fliM mutations; this is consistent with the idea that the CheY protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.

In Escherichia coli and Salmonella typhimurium, chemo- We have used genetic suppression, a useful tool for taxis is achieved by regulating the direction of flagellar investigation of protein interactions (for general reviews, see rotation (reviewed in reference 46). Flagellar rotation is references 7, 14, and 18; for specific examples, see refer- controlled by the flagellar motor switch apparatus, a multi- ences 1, 2, 12, 13, 19, 21, 23, 26, 29, 30, 32, 43, 49, and 50), component macromolecular structure composed of at least to study structure-function aspects of CheY activity at the three different protein subunits, FliG, FliM, and FliN (55, switch. A number of earlier suppression studies have dem- 56), which imparts either clockwise (CW) or counterclock- onstrated interactions between cheY and flagellar switch wise (CCW) rotation to the motor. CW flagellar rotation genes fliG, fliM, and fliN (37, 38, 44, 55). In this study, we results in a tumble in the swimming pattern, while CCW extended the genetics of the CheY-switch interaction to the rotation results in smooth swimming. By continually sam- mapping of dominant suppressors of flagellar switch muta- pling the environment over time and modulating flagellar tions on the molecular structure of CheY. The amino acid rotation accordingly, cells can move toward a more favor- residues shown here to be critical for CheY signaling cluster able, and away from a less favorable, environment. on one face of the CheY protein, suggesting that this region The (Che) proteins couple flagellar rotation to of CheY transduces chemotactic signals directly to the the environment by transducing chemotactic signals from switch. specific transmembrane chemoreceptors to the flagellar switch apparatus. One of these proteins, CheY, is a small (14-kDa), single-domain protein homologous to the regulator MATERIALS AND METHODS proteins of bacterial two-component sensory transduction systems (3) and is the only two-component signaling protein Reagents. Restriction enzymes and T4 DNA ligase were for which the high-resolution crystal structure has been purchased from Bethesda Research Laboratories (Gaithers- determined (47, 51). Like other regulators, CheY activity burg, Md.). Bacto-Tryptone, Bacto-Agar, and Bacto-Yeast appears to be controlled by phosphorylation (reviewed in Extract were from Difco Laboratories (Detroit, Mich.). Calf references 8 and 48); cheY function is required for CW intestinal alkaline phosphatase, used in some cloning exper- flagellar rotation (34, 35); CheY is phosphorylated in vitro by iments, was obtained from Boehringer Mannheim Biochem- CheA (16, 17, 33, 54); and cheY or cheA mutations that icals (Indianapolis, Ind.). Molecular biology grade agarose disrupt phosphotransfer reactions also result in smooth- was from International Biotechnologies, Inc. (New Haven, swimming phenotypes, suggesting that CheY-P is the active Conn.). Penicillin, hydroxylamine hydrochloride, protein CW generator (9, 33). While CheY is thought to act directly A-agarose, cyanogen bromide-activated Sepharose 4B, and on the switch apparatus to regulate flagellar rotation (11, 38, 3-3-indoleacrylic acid were obtained from Sigma Chemical 39, 53, 55), exactly how CheY functions is not known. Co. (St. Louis, Mo.). 125I-labeled protein A was purchased from ICN Biomedicals, Inc. (Costa Mesa, Calif.), and * Corresponding author. [y-32P]ATP was from Amersham Corp. (Arlington Heights, t Present address: Department of Biochemistry, Molecular Biol- Ill.). Other specialized enzymes and reagents are noted ogy and Cell Biology, Northwestern University, Evanston, IL where applicable. All other reagents and chemicals were of 60208. reagent grade and were obtained from Sigma Chemical Co., 6247 6248 ROMAN ET AL. J. BACT1ERIOL.

TABLE 1. Bacterial strains and plasmids Strain or plasmid Characteristic(s) Source (reference); Strains RP437 Wild type (Che+) J. S. Parkinson RP4139 cheZ280 recA J. S. Parkinson RP4079 cheY216 recA J. S. Parkinson RP4650 cheB270 J. S. Parkinson RP5135 Atar-cheZ J. S. Parkinson RP4500 fliG1009 cheY+ J. S. Parkinson' RP4501 fliGlOlO cheY+ J. S. Parkinson' RP4503 fliGlO12 cheY+ J. S. Parkinson' RP4504 fliG1013 cheY+ J. S. Parkinson' RP4505 fliG1014 cheY+ J. S. Parkinson' RP4520 fliGlO29 cheY+ J. S. Parkinson' RP4496 fliMi005 cheY+ J. S. Parkinson RP4506 fliM1015 cheY+ J. S. Parkinson' RP4510 fliM1019 cheY+ J. S. Parkinson" RP4511 fliMi020 cheY+ J. S. Parkinson RP4517 fliM1026 cheYr J. S. Parkinson' AZ37 fliGlO37 cheYr This workb AZ38 fliGlO38 cheY+ This work" AZ39 fliG1O39 cheY+ This work' Plasmids pFZY oriF (low copy number) Penr A. Koop (20) via M. Winkler pMM1 tar operon (tar tap cheR cheB cheY cheZ flhB') in pFZY M. Meyers pRL22 Overproduces CheY and CheZ from tqp promoter R. Linzmeier pRL22AYC cheY deletion, overproduces CheZ only D. Vacante pRL22AZd cheZ deletion, overproduces CheY only This work pDV4 Overproduces CheA and CheW from trp promoter D. Vacante pYBO902 pMM1 cheY0902[T112I] This work pYBO903 pMM1 cheYO903[A9OV] This work pYBO904 pMM1 cheY0904[E117K] This work pYBO905 pMM1 cheYO905[E117K] This work pYBO906 pMM1 cheY0906[V108M] This work pYB1304 pMMl cheY1304[V11M] This work pYB1308 pMM1 cheY1308[T1121] This work pYB1409 pMM1 cheY1409[A9OT] Tlhis work pYB1610 pMM1 cheY1610[F111V] This work' pYB2885 pMM1 cheY2885[E27K] This worke pRYBO902d pRL22 cheY0902[T112I] This work pRYBO903d pRL22 cheYO903[A9OV] This work pRYBO904d pRL22 cheY0904[E117K] This work pRYBo9O5d pRL22 cheYO905[E117K] This work pRYBO906d pRL22 cheY0906[V108M] This work pRYB1304d pRL22 cheY1304[V11M] This work pRYB1308d pRL22 cheYl308[T112I] This work pRYB1409d pRL22 cheY1409[A90T] This work pRYB1610d pRL22 cheY1610[F111V] This work pRYB2885d pRL22 cheY2885[E27K] This work pRBB40.cheYD13K pRL22 derivative R. Bourret pRBB40.cheYD13KAZ AcheZ derivative This work pRBB40.cheYD57N pRL22 derivative R. Bourret pRBB40.cheYD57NAZ AcheZ derivative This work

a J. S. Parkinson's original fliG (scyB) and fliM (scyA) allele numbers are retained as the last digits of our allele numbers. For example, fliGl100 = scyB1O, fliM1005 = scyAS, etc. b These strains were constructed in our laboratory from spontaneous chromosomal fliG suppressors of cheY304[Vl1M]. C pRL224Y, which was used to express CheZ, was produced by ExolIl-Sl digestion from the Sall site in cheY. In this construction, approximately 51 bp are deleted from cheY, resulting in an in-frame deletion that yields an internally deleted, nonfunctional CheY peptide (50a). d AZ versions of all high-copy-number cheY expression plasmids (both cheY+ and suppressor cheY alleles) were produced by PvuII deletion of cheZ. These plasmids were used to overproduce wild-type and suppressor CheY proteins in the absence of CheZ. The original strains with these cheY alleles are from J. S. Parkinson. The cheY alleles were cloned from the chromosome with mini-Mu and then subcloned into M13 and sequenced (5). To characterize dominant suppression, the cheY alleles were subcloned from mini-Mu into pMM1.

Aldrich Chemical Co. (Milwaukee, Wis.), or Fisher Scien- tonA31 tsx-78), a Che+ reference strain that is itself a tific Co. (Fair Lawn, N.J.). derivative of E. coli K-12 (36). Switch mutant tester strains Bacterial strains and plasmids. Strains used in this study are Mot' Che- (motile but nonchemotactis), genotypically are listed with their relevant genotypes in Table 1. All strains denotedfliG cheY+ orfliM cheY+. They containfliG orfliM are derivatives of RP437 (F- thr-l(Amn) leuB6 his-4 alleles that are themselves suppressors of cheY mutations metF159(Am) eda-SO rpsL136 thi-I ara-14 lacYl mtl-l xyl-S and that have been crossed back into wild-type cells (38). VOL. 174, 1992 CheY SIGNALING SURFACE 6249

These tester strains lack the suppressible mutant cheY phosphorylation buffer (28). Unincorporated label was alleles and are consequently Che-. Several otherfliG cheYr washed away with phosphorylation buffer, and the immuno- tester strains (AZ strains) were generated in our laboratory precipitated, labeled complexes were distributed into 25 to from spontaneous fliG suppressors of cheY1304[V11M]. 50 fresh tubes. Phosphotransfer reactions were initiated by All of the plasmids used in this study are described in addition of 20 ,ul of an appropriate mixture of different Table 1. lysates to an aliquot of labeled CheA-CheW. The reactions Bacterial growth conditions. L broth, L agar (40), and contained wild-type or mutant CheY (35 pmol) with or tryptone swarm agar (34) have been previously described. without CheZ (35 pmol), plus control lysate (no CheY or Penicillin was used at 100 ,ug/ml when required for plasmid CheZ) and phosphorylation buffer to bring the reaction to 35 maintenance. Swarm plate assays for chemotaxis (34) were ,ug 9f total protein. Reactions were terminated at 5, 10, 20, performed at 30°C for up to 14 h. All other cell growth was and 40 s by addition of an equal volume of 2x sodium at 37°C. Induction of che gene expression from the trp dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- promoter of pRL22 (and its derivatives) or pDV4 was PAGE) sample buffer containing 20 mM EDTA, and 10 to 20 accomplished by addition of 3-,B-indoleacrylic acid to a final ,ul of this material was loaded directly onto SDS-15% PAGE concentration of 100 ,ug/ml to mid-log-phase L-broth-grown gels (no boiling). Radiolabeled proteins were visualized by cultures. autoradiography, and radioactivity in protein bands was In vitro mutagenesis and selection of suppressor mutations. determined with an Ambis ,3-scanning system. Approxi- Low-copy-number tar operon plasmid pMM1 was randomly mately 11 pmol of CheA and 14 pmol of CheW were present mutagenized in vitro with hydroxylamine by a published in phosphotransfer reactions, although some variation in the procedure (33). Transformant colonies offliG cheY+ strains amounts of immunoprecipitated proteins was observed. containing mutagenized pMM1 were pooled, and aliquots CheY cross-linking. CheA was purified by published pro- were inoculated in troughs through the centers of swarm cedures (16) and covalently bound to cyanogen bromide- agar plates containing penicillin. Che+ transformants, rec- activated Sepharose 4B. Aliquots of the CheA resin corre- ognized by rapid swarming away from the point of inocula- sponding to 10 ,ug (130 pmol) of CheA were distributed to tion, were isolated, and putative suppressor mutation-bear- polypropylene minispin columns and washed with MKM (25 ing plasmids were purified and analyzed by restriction mM MOPS [pH 7.9], 50 mM KCl, 5 mM MgClj2. Autophos- enzyme digestion. These plasmids were used both to retrans- phorylation of CheA was allowed to proceed for 20 min at form the original fliG cheY+ strain (to confirm that the room temperature in 50-,u reactions containing 10 to 100 ,uCi suppression was plasmid encoded) and for complementation of [.y-32P]ATP and 0 to 100 ,uM cold ATP in MKM. Excess assays in strains RP4079 (cheY216) and RP4139 (cheZ280). ATP was washed away with MKM, and phosphotransfer Only one isolate was saved from any single transformation reactions were initiated by addition of 50 pl (80 pmol) of pool to avoid isolation of siblings. purified wild-type CheY (27) in MKM. After 10 s, the Subcloning and DNA sequencing. High-copy-number ex- products of each reaction were centrifuged directly into 2 pl pression plasmids bearing putative suppressor cheY alleles of 5 mM disuccinimidyl suberate (a covalent cross-linking were created by subcloning the 2.4-kb HindIII-BamHI cheY- reagent from Pierce Chemical Co.) in dimethyl sulfoxide. cheZ fragment from low-copy-number suppressor plasmids Cross-linking was allowed to proceed for 5 s, and 50 RI of 2x into pRL22 (27). Constructions were confirmed by comple- SDS-PAGE sample buffer containing 20 mM EDTA was mentation assays and restriction digestions. added to terminate each reaction. Products of the cross- DNA sequencing of double-stranded high- and low-copy- linking reactions were visualized by autoradiography follow- number plasmids was done by the chain termination method ing SDS-PAGE in 15% gels. Radioactivity in protein bands (42) using Sequenase (U.S. Biochemical Corp., Cleveland, was determined with an Ambis 3-scanning system. Western Ohio). Three appropriately situated oligonucleotide primers blots were also performed on parallel unlabeled reactions to specific for cheY (noncoding strand) facilitated sequencing. determine the total CheY in monomers and multimers. Analysis of allele specificity. Low-copy-number plasmids bearing suppressor cheY mutations were transformed into RESULTS fliG cheY+ and fliM cheY+ strains and tested for restoration of chemotaxis by swarm assay. Final swarm size was Selection of dominant cheY suppressors offliG mutations. determined for nine isolates of each plasmid-strain combina- Our genetic selection was designed to recover dominant tion to yield mean values relative to wild-type controls cheY suppressors of fliG mutations. Low-copy-number tar (RP437 containing pMM1). operon plasmid pMM1 was randomly mutagenized in vitro Phosphorylation biochemistry of suppressor CheY proteins. and transformed into various fiG cheY+ tester strains. Che proteins were overproduced in Atar operon strain Transformants were tested for restoration of swarming on RP5135, and overproducing cultures were sonicated in phos- semisolid agar plates. This procedure yielded 15 suppressor phorylation buffer (50 mM Tris [pH 7.9], 50 mM KCl, 5 mM isolates; 8 had mutations in cheY, and 7 had mutations in MgCl2). Total protein in lysates was determined by using a cheZ. The generation of both cheY and cheZ suppressors of Coomassie-based dye-binding reaction (Pierce Chemical switch mutations is consistent with earlier studies that Co., Rockford, Ill.). Quantitative Western blots (immuno- demonstrated genetic interaction between cheY, cheZ, and blots) with anti-Che protein antibodies and "25I-labeled pro- switch genes (37, 38, 44, 55). In addition to the cheY alleles tein A were done to determine the amounts of Che proteins we generated, two previously characterized (38) switch in crude lysates and immunoprecipitates. Radioactivity in suppressor cheYalleles (Table 1) were subcloned into pMM1 immunoreactive protein bands was determined with an Am- and shown to have dominant suppression activity in our bis 1-scanning system. system. Dominant effects in our system are not a result of CheA-CheW complexes were immunoprecipitated from overproduction of the mutant protein product, since cheY crude CheA-CheW overproduction lysates with anti-CheA expression is controlled by its native promoter on a vector antibody and protein A-agarose beads, and CheA was auto- that is maintained at one or two copies in the cell (20). phosphorylated with 50 to 100 ,uCi of [,y-32P]ATP for 5 min in Suppressor cheY alleles were subcloned into high-copy- 6250 ROMAN ET AL. J. BACTERIOL. -fiM 005 sive protein interaction between CheY and FliG or FliM. cheY2885 [E27K]* Conformational suppressors might exhibit low specificity if FcheYO902 [T112I] the nature of the protein interaction is complex (e.g., multi- cheY216 -fliG1009 -cheY0903 [A9O0V protein complexes) and/or the assay for suppression is [E27K -cheYO904 [E117KI indirect. Alternatively, these results may indicate that sup- -cheYO905 [E117K] pression occurs by a less direct mechanism (i.e., not directly involving specific restoration of a disrupted CheY-FliG or -cheY0906 [V1O8M] CheY-FliM interaction). We explore these possibilities fur- - *i(101fl -cheiY1R6lo rFll1lV1* fltiG1037 ther in the Discussion. [VllMl In vitro phosphorylation biochemistry of suppressor CheY fliGlOl3 j-cheY1304 -fliG1038 proteins. CheY is a multifunctional protein that interacts FXII101 '-cheYl3O8 [T112I] -fliG1039 with a number of other Che and motility-chemotaxis-related cheY220 fliG1014 -cheY1409 [A9OT] proteins. Therefore, we undertook a biochemical character- [S56F] ization of wild-type and suppressor CheY protein functions L-fliM1015 to investigate the extent to which other CheY interactions and biochemical properties could account for suppressor activity. cheY219- One critical component of CheY function is the ability to fliM1020 be phosphorylated by CheA and dephosphorylated by CheZ. We have been able, by assaying the phosphorylation bio- fliM1026 chemistry of CheY proteins in vitro, to distinguish phospho- cheY201- rylation-competent from phosphorylation-defective pro- -fliG1029 teins. Our in vitro phosphorylation assay, based on reactions FIG. 1. Lineage of cheY suppressors of flagellar switch muta- using purified Che proteins (16, 17, 54), utilized lysates tions. cheY, fiG, and fliM allele numbers and inferred amino acid containing overproduced Che proteins. The assay allowed us changes (for sequenced cheY alleles) are shown. cheY alleles to characterize the transfer of phosphate from CheA to originally isolated as spontaneous chromosomal suppressors offliG CheY and the subsequent removal of phosphate from CheY mutations are marked with asterisks. Three new fiG cheY+ strains by CheZ. Examples of the results obtained from such assays generated by spontaneous reversion of cheY1304[V11M] are on the are presented in Fig. 3, and Table 2 summarizes the results far right. for all of the CheY proteins assayed. All of the suppressor CheY proteins, except the V11M protein, were qualitatively the same as wild-type CheY in our assay. This indicates that number expression plasmid pRL22 (27) and sequenced. the alterations that underlie their suppressor activity did not Some suppressor cheY alleles were sequenced from low- result in any defects in phosphorylation biochemistry (which copy-number plasmid DNA prior to subcloning. The CheY depends on both the chemistry of phosphotransfer reactions amino acid substitutions inferred from these mutations are and interaction with CheA and CheZ). Two other CheY listed in Fig. 1, along with their lineages. mutants, D13K and D57N, were used as controls to ensure Analysis of suppressor CheY allele specificity. One mecha- that our assay would reliably distinguish between phospho- nism by which intergenic suppression can occur is confor- rylation-competent and defective CheY proteins. These mational suppression. If two proteins make stereospecific CheY proteins, previously assayed in purified form and contact, then mutations in one protein that disrupt this shown to be defective in transfer of phosphate from C-heA interaction may be suppressed by compensating mutations in (9), were clearly phosphorylation defective in our assay. the other protein that restore the interaction. Consequently, CheY cross-linking. Another potential mechanism that the amino acid residues identified by conformational sup- might account for suppressor CheY function is an alteration pression should define regions of physical contact between of CheY self-association. OmpR, the porin gene transcrip- proteins because they contribute to the topographies of tion factor, is a two-component regulator protein homolo- protein interaction surfaces. Conformational suppressors gous to CheY for which phosphorylation-dependent dimer- should also be highly allele specific, since they reflect the ization has been demonstrated (31). Interestingly, two ompR precise stereospecific contact between interacting proteins. mutations whose products exhibit normal phosphorylation To assess the allele specificity of our suppressors, we biochemistry but fail to dimerize and bind DNA have been tested their ability to restore swarming in different fliG characterized (31). These mutations affect residues of OmpR cheY' backgrounds. Our cheY alleles showed a low level of that are, by sequence alignment, close to some of the specificity when tested in eightfliG cheY' tester strains (Fig. residues we have identified by suppression. This raised the 2A). The level of specificity was not as high as might be possibility that these residues could be involved in a similar expected for a direct, exclusive one-to-one interaction of function for CheY activity. CheY with FliG. We assayed wild-type CheY multimer formation over a cheY mutations can be specifically suppressed by muta- range of CheY-P concentrations (0 to 15 nM) by using the tions in either fliG or fliM, so we also tested our suppressor rapid chemical cross-linking reagent disuccinimidyl suber- cheY alleles in five fliM cheY' strains (Fig. 2B). Surpris- ate. The fraction of CheY-P (or CheY) in multimers did not ingly, the patterns of suppression by cheY alleles of bothfliG increase with increasing CheY-P concentrations, as deter- and fliM mutations were similar and the greatest swarming mined both by quantitation of radiolabeled CheY-P and by actually occurred in several of the fliM strains. This was not Western blotting (data not shown). Thus, phosphorylation expected, since our suppressor cheY alleles were generated had no detectable effect on CheY self-association, implying against fliG, not fliM, mutations. that CheY-CheY interaction is not required for CheY func- The lack of high specificity for alleles of fliG and the tion. similar suppression offliM alleles do not disprove an exclu- Mapping of suppressor CheY mutations on the CheY struc- VOL. 174, 1992 CheY SIGNALING SURFACE 6251

A.50 I I I I I I I I I 125 fliG1014 100 75 - tw - sw -*n--- w - '.3v- --.If I cosC 501 X a-I 25 0a1. 4- 12 : ,liG1029 fliG1037 fliGl038 tliGI039 1a20O

~Al~k±+ 4 o0 , . A . r . . I --." .'~~~~~~~~~~~~~~~~~~~~~\K 41.1 a+ 0 & & ZA -A, -il 41!. .+ -., A .? .r Ai? t A ,,. & 4'v 4 tv ; 1. 4y .p e "q, Al. '4.4 le '4.4 4.*, .?

I I v I I I I I I B 125 tiM005 . tziml6i tTimloig, .*__ fl~~~~~~~~~tiM1020 100 II I _', I! !_,x > >-r >> § 75 II 250 _.A-

10

25

FIG. 2. Analysis of suppressor cheY allele specificity. Low-copy-number plasmids bearing suppressor cheY alleles were transformed into various fliG cheY+ (A) and fliM cheYr (B) switch mutant tester strains and assayed for swarming on semisolid agar plates (34). Each panel represents the suppression profile in a given tester strain. Suppressor cheY alleles are ordered from best to worst along the x axis with respect to suppression offliG1O29, with which the greatest level offliG suppression was observed. Suppression datum points (0) are connected for ease of comparison only. Dashed horizontal lines represent the residual swarming behavior of the fli cheY+ tester strains. Crosses mark the swarming behaviors that resulted when suppressor cheY alleles were introduced into the wild-type reference strain (RP437). Thus, the shaded regions represent the ranges of swarm sizes that fall between the swarm phenotypes observed for single-component (fli or cheY) mutations in an otherwise wild-type background. Datum points that fall above these shaded regions represent suppressor-mutant combinations with greater swarming ability than either single-component mutation by itself. ture. Figure 4 depicts the CheY amino acid residues we have chains also extend out to the surrounding solvent, toward a identified as being critical for signaling on the molecular region where the switch proteins might be located in the structure of E. coli CheY. The location of these residues is CheY-switch interaction. Clearly, substitutions at these sites striking-they cluster to a particular region of CheY and are could change the topography of the proposed signaling located on the surface of the protein (except Vii). This surface; this is consistent with the possibility that this strong clustering suggests a functional role for this region of surface functions in protein interaction. The substitutions we the CheY molecule, and we propose that it is a signaling observed make chemical sense in the context of an altered surface-a distinct part of CheY that functions in transduc- protein interaction surface. For example, the two glutamate- tion of chemotactic signals. This proposed signaling surface to-lysine substitutions (E27K and E117K) are charge consists of solvent-accessible surfaces of the C-terminal changes that might be expected to affect salt bridges with portion of a-i (E27), the loop region between ,-4 and a-4 interacting proteins. The other substitutions involve side- (A90), the loop between ,-5 and a-5 (V108, Flll, and T112), group volume changes (A9OV and V108M), as well as and a-5 (E117). hydrophobicity changes (A9OT and T112I). Of the seven Not only are all of the residues except two (Vll and Flll) surface residues, only Flll has its side chain directed in surface located and hence solvent accessible, but their side toward the hydrophobic core of CheY. In this case, only the 6252 ROMAN ET AL. J. BAcTERiOL.

WT A90V ChicY lr ineY, (Ihez ChuY aione ChE.Ch / 5 - rJ .{: 5 5 C -T' 4 5 10 -, 10 0 me isec 12i q.4; ..

*. CheA-P I.-..... *-- -eA-P

4- ChleY-P _ 4 CheY-P 4w:~~~~~~~~~~~~~~~~~~~~~~~~ ,--.-- -" o- - 4wmo:mw D13K VllM CheY alone CheY +CheZ ClheY a'ore C.eY-ChecZ 0 5 10 20 40 5 10 20 40 lime (sec) lr5 1 C Time (sec)

k-P -4 _-',heA-P

.._ CheY-P

.. i:.

FIG. 3. Phosphorylation properties of wild-type and mutant FIG. 4. Stereograph showing the locations of suppressor cheY CheY proteins. After phosphotransfer reactions (see Materials and mutation sites on the 1.7-A CheY structure (51). Highlighted resi- Methods), samples were analyzed by SDS-PAGE and autoradiog- dues are as follows: blue atoms represent residues identified by raphy. The reactions for only one CheY species are shown in a given suppression analysis (including parental mutation S56F), red atoms autoradiogram (labeled above each panel). Differences in band represent phosphorylation active-site residues (D12, D13, and D57), intensity are due to differences in film exposure and the specific and white atoms represent K109 (see text for a discussion). The activity of [y-32P]ATP used for initial labeling of CheA. The total a-carbon backbone of CheY is in purple. Calculated solvent-acces- label loaded in each lane of a given gel was the same for all lanes, sible surfaces of the highlighted residues are depicted by white except for the 0-s (no CheY added) lane for wild-type CheY, which stippling. The viewpoint of this diagram is over the active-site region contained about twice as much label as the other lanes of that gel. of CheY.

DISCUSSION backbone portion of this residue seems to contribute to the proposed signaling surface, and the influence of the side- Genetic suppression has defined residues of CheY critical chain substitution may be more indirect. Vii, since it is in for its signaling function at the switch apparatus. Our goal in the hydrophobic core of CheY, is not part of this surface. this study was to use a genetic approach to investigate structure-function aspects of CheY signaling at the flagellar switch. A number of genetic studies have indicated interac- TABLE 2. Phosphorylation phenotypes of wild-type and tions between CheY and components of the switch (37, 38, suppressor CheY proteinsa 44, 55). To extend these studies, we have generated and characterized a set of cheY mutations that dominantly sup- Phosphorylation phenotype % as CheA-P % as CheY-P press flagellar switch mutations. and protein Dominant mutants are readily understandable in terms of Wild protein-protein interactions and have been interpreted as Wild type 0.9 67.4 evidence of functional contacts between proteins. Dominant E27K 1.4 31.2 mutants can be explained in terms of spoiling and competing A9OV 0.8 42.8 (23). Dominant spoilers are loss-of-function mutants that A90T 0.4 76.2 inactivate the wild-type counterpart, usually through het- V108M 1.1 69.1 erodimer formation. These types of mutants, sometines F111V 0.9 34.0 called dominant negative mutants, have recently been used T112I 1.4 40.3 E117K 1.6 84.5 to demonstrate protein interactions (4, 15). Dominant com- petitors are gain-of-function mutants that override the func- Defective tion of the wild-type counterpart, usually through interac- VllM 51.6 6.2 tions with other components. Dominant competitors are also D13K 77.6 11.4 useful for determination of protein interactions, as they may D57N 80.8 10.8 be indicative of altered protein affinities (23). We reasoned a Phosphorylation wild-type CheY proteins removed >98% of the label that by requiring dominance we could favor the isolation of from CheA, while phosphorylation-defective CheY proteins removed <50% suppressors that function by affecting protein interactions. of the label from CheA (by the end of the assay). In addition, wild-type The residues we have defined by suppression cluster on proteins showed maximum labeling of >30% while defective proteins showed the molecular structure of CheY and are critical to the CheY maximum labeling of <10% of the label originally associated with CheA (usualiy at the 5-s time point). All phospho-CheY proteins were sensitive to protein's ability to transduce chemotactic signals. While we CheZ dephosphorylation, generally retaining < 1% of the total label by the end cannot conclusively state that these residues function di- of the assay when CheZ was present. The data are percentages of the total rectly in switch binding, we propose that they contribute to label in the reaction detected as CheA-P or CheY-P and are from a single a signaling surface on CheY that functions either directly in representative assay (except for wild-type CheY, for which the values are averages of five assays). Assays of all CheY proteins were done several times binding to the switch or in some postbinding activity re- and always gave qualitatively similar results, but because of variation in the quired for signaling. In either case, since substituted sup- assay conditions, the results are not quantitatively comparable. pressor proteins were wild type for other known CheY VOL. 174, 1992 CheY SIGNALING SURFACE 6253 functions, the functional target for the signaling activity of the residues in this study (44). It may require additional this surface appears to be the flagellar switch. mutagenic procedures to define this functional surface of We are aware of two other cases in which suppressor CheY completely. mutations have been mapped on protein molecular struc- A multidiscipline approach to protein function. Genetic tures: for maltose-binding protein from E. coli (45, 57) and analysis, particularly the paradigm of conformational sup- for CheY from S. typhimurium (44). In reference 44, the pression, is a powerful and valuable method for investigation positions of nine chromosomal cheY suppressors were of protein interactions. Indeed, there is no quicker and easier mapped on the CheY structure. Six of these suppressors approach to the generation of a host of valuable mutants for mapped within or close to our proposed signaling surface. Of the study of protein function, but genetics, being an inher- the other three, one or two may be defective for phosphor- ently indirect approach, has limitations that must be consid- ylation (this was not determined but is inferred from their ered in any system. The extent to which genetics can clearly locations), and the last suppressor mapped on a region of and reliably determine protein interactions depends on the CheY opposite our proposed signaling surface. These results degree to which the phenotype reflects the restoration of a are remarkably consistent with our results, considering the protein interaction (i.e., the directness of the phenotypic different selection protocols used. The tighter clustering of assay), as well as the nature of the interaction (i.e., a suppressor mutations we recovered may be due to the minimal number of interacting components). However, the additional requirement of dominance in our screening proto- ability to combine genetics with biochemical and structural col. analyses can provide a much more complete picture of Signaling surface and the mechanism of CheY activation. protein function. Figure 4 shows the proposed signaling surface on the molec- The CheY-switch system illustrates some limitations of a ular structure of CheY. The location of the signaling surface purely genetic approach to the investigation of protein correlates well with mechanistic models put forth for CheY interactions. Swarm assays, while convenient and easy to activation (24, 51). In the nonphosphorylated form of CheY, perform, are indirect. Swarming is a result of numerous an unusual linkage (cis) between K109 and P110 allows the factors, including CheY activation via phosphorylation and e-amino group of K109 to bind to a carboxylate oxygen of the ability of activated CheY to interact with and have an D57, 2.7 A away (51). The K109-D57 bond is expected to be effect on the switch, and even less direct factors, like the quite stable in solution because of its buried location and the growth rate and metabolic state of the culture. Wolfe and extremely low solvent accessibility of the K109 and D57 side Berg (52) showed that some Che- strains are able to swarm, chains (51). According to recent protein structure analysis, a and swarm size is affected by the intrinsic CW-CCW rota- side chain is considered buried if the accessible surface area tional bias of the switch. These mutants have at least is less than 40 A2 (10), and K109 and D57 have solvent- partially bypassed the requirement for chemotactic signaling accessible surfaces of 38.5 and 2.5 A2, respectively (51). to form swarms. Similar results were reported by Magar- Formation of the K109-D57 bond constrains the C-terminal iyama et al. (25) and Sockett et al. (44) after extensive portion of the protein (specifically, a-5 and the loop between genetic analysis of spontaneous switch gene suppressors of a-5 and 1-5) in a particular conformation. Phosphorylation of cheY mutations in S. typhimurium. These investigators the carboxyl group on D57 would affect the K109-D57 concluded that suppression by most of their spontaneous association, consequently altering the conformation of a-5 switch mutations could be explained by an altered intrinsic and the a-5-1-5 loop (24, 51). This portion of CheY makes up CW-CCW bias that compensated for weaker binding by the central part of our proposed signaling surface. Our mutant CheY proteins rather than restoration of normal (or hypothesis is that phosphorylation activates CheY through a nearly normal) binding. Sockett et al. (44) conceded that true conformational change of the signaling surface, which re- conformational suppression probably does occur in the sults in either increased affinity for the flagellar switch or CheY-flagellar switch interaction, but is difficult to distin- increased ability to communicate a signal to the switch once guish against the background from the more general bias CheY is bound. correction phenomenon. They have used the term "pseudo- Figure 4 also reveals that the signaling surface is distinct taxis" to refer to such chemotaxis-independent swarming. from, but contiguous with, the active site of phosphorylation The swarm phenotypes associated with the Vl1M protein defined by residues D12, D13, and D57 (9, 41, 51). This, may be due to pseudotaxis, since the V11M protein is together with the finding that all of the suppressor mutants phosphoxylation defective and the normal pathway of (except V11M) exhibited normal phosphorylation biochem- chemotactic should be interrupted at this istry, is consistent with the idea, first suggested by Bourret step. The V11M protein is a different kind of mutant on the et al. (9), that phosphorylation and switch binding-induction basis of its location and phosphorylation properties, and of CW rotation are functionally and physically separable. suppression (restoration of swarming) probably occurs by a Despite the recurrence of suppressor mutations T112I and different mechanism. E117K, this region of the CheY molecule does not appear to The complex structure and function of the target of CheY be mutationally saturated. We have analyzed only a limited activity, the flagellar switch apparatus, also imposes a limi- number of suppressor mutations, and several residues not tation on the genetics of this system. As discussed above, identified in this study seem to be good positions for sup- switch mutations can suppress cheY mutations by mecha- pressor mutations in CheY. These residues are bounded by, nisms that presumably do not involve restoration of the or very close to, the signaling surface defined by suppressor CheY-switch interaction. In addition, the switch is com- mutations and have side chains directed out to the surround- posed of several protein subunits, and both our data and ing solvent medium. Examples of such residues include L24 those of others (37, 38, 44, 55) indicate that CheY may (on the loop between 3-1 and a-1), L28 (a-1), A113, A114, interact with any or all of the switch proteins. Such a target E118, and K119 (all on a-5). There is no a priori reason why may present a complex binding site for CheY. Thus, whereas these positions were not identified by mutations in our study. allele specificity should be a hallmark of conformational In fact, the CheY residues similarly identified by suppression suppression, the nature of the protein interactions and the in S. typhimurium that map in this region are different from complex function of the switch may have contributed to the 6254 ROMAN ET AL. J. BAcTERIOL.

lack of specificity we have observed in the CheY-switch Vacante for constructing pRL22AY, A. van der Zel for constructing system. the AZfliG tester strains, R. Bourret for plasmids that express CheY Mechanism of suppression and function of the signaling mutants D13K and D57N, H. Wang for developing CheA-Sepharose surface. Since our data do not allow us to for phosphotransfers to CheY, and J. S. Parkinson for generously define clearly the providing strains. mechanism of suppression by mutant CheY proteins, we can This work was supported by NIH Public Health Service grants AI only speculate on the function of the proposed signaling 18985 (P.M.), GM 39919 (K.V.), and GM 13662 (S.R.). surface during chemotactic signaling. If CheY binding to the switch is both necessary and sufficient for switching (a REFERENCES single-step switching model), then the signaling surface must 1. Adams, A. E. M., and D. Botstein. 1989. Dominant suppressors function directly as a switch-binding surface. If CheY bind- of yeast actin mutations that are reciprocally suppressed. Ge- ing to the switch is necessary but not sufficient for switching netics 121:675-683. (a multistep switching model), then the signaling surface 2. Adams, A. E. M., D. Botstein, and D. G. Drubin. 1989. A yeast could either function as the switch-binding surface or have actin-binding protein is encoded by SAC6, a gene found by some required signaling function subsequent to binding. suppression of an actin mutation. Science 243:231-233. Models for switching that could accommodate either of these 3. Albright, L. M., E. Huala, and F. M. Ausubel. 1989. Prokaryotic scenarios have been signal transduction mediated by sensor and regulator protein presented (6, 22). Alternatively, muta- pairs. Annu. Rev. Genet. 23:311-336. tions of the residues we have identified could somehow 4. Amaya, E., T. J. Musci, and M. W. Kirschner. 1991. Expression indirectly influence a separate region of CheY involved in of a dominant negative mutant of the FGF receptor disrupts switch binding or a subsequent signaling function. While this mesoderm formation in Xenopus embryos. Cell 66:257-270. possibility seems less likely, it cannot be formally excluded. 5. Beman, J. 1987. Examination of the interaction between chemo- The nature of the switch defects obviously dictates the taxis and flagellar gene products in Eschenichia coli: cloning and possible mechanisms of suppression by mutant CheY pro- sequence analysis of cheY mutants. M.S. thesis. University of teins. Since the switch mutant strains we used have pro- Illinois, Chicago. nounced CW rotational biases in the presence of wild- 6. Block, S. M., J. E. Segall, and H. C. Berg. 1983. Adaptation CheY not be for kinetics in bacterial chemotaxis. J. Bacteriol. 154:312-323. type (38), they may specifically impaired 7. Botstein, D., and R. Maurer. 1982. Genetic approaches to the interaction with CheY. In such cases, the suppression we analysis of microbial development. Annu. Rev. Genet. 16:61- observed would not result from specific restoration of a 83. disrupted CheY-switch interaction, as in classical conforma- 8. Bourret, R. B., K. A. Borkovich, and M. I. Simon. 1991. Signal tional suppression. The defects of these mutant switches transduction pathways involving protein phosphorylation in might be altered intrinsic CW-CCW biases or altered sensi- prokaryotes. Annu. Rev. Biochem. 60:401-441. tivities to chemotactic signals, and suppression could be 9. Bourret, R. B., J. F. Hess, and M. I. Simon. 1990. Conserved explained by CheY mutants that effectively reset or over- aspartate residues and phosphorylation in signal transduction by come the altered bias or sensitivity of a mutant flagellar the chemotaxis protein CheY. Proc. Natl. Acad. Sci. USA switch. 87:41-45. 10. Bowie, J. U., R. Liithy, and D. Eisenberg. 1991. A method to We can explain the dominant suppression by our CheY identify protein sequences that fold into a known three-dimen- mutants in terms of competition (gain of function) and sional structure. Science 253:164-170. spoiling (loss of function). As competitors, our CheY mu- 11. Clegg, D. O., and D. E. Koshland, Jr. 1984. The role of a tants may be true conformational suppressors or they may signaling protein in bacterial sensing: behavioral effects of act more generally to reset the bias or sensitivity of mutant increased gene expression. Proc. Natl. Acad. Sci. USA 81: switches. The latter types of suppressors include mutants 5056-5060. that correct a less sensitive switch by communicating stron- 12. Das, A., C. Merril, and S. Adhya. 1978. Interaction of RNA ger, properly integrated signals and mutants that reset rota- polymerase and rho in transcription termination: coupled AT- tional bias virtue of an enhanced Pase. Proc. Natl. Acad. Sci. USA 75:4828-4832. by CCW-promoting func- 13. Guarente, L., and J. Beckwith. 1978. Mutant RNA polymerase tion. As spoilers, CheY suppressors might reset a mutant of Eschenichia coli terminates transcription in strains making switch bias by acting as phosphate sinks that accept phos- defective rho factor. Proc. Natl. Acad. Sci. USA 75:294-297. phoryl groups but fail to communicate CW signals to the 14. Hartman, P. E., and J. R. Roth. 1973. Mechanisms of suppres- switch. Only true conformational suppressors would be sion. Adv. Genet. 17:1-105. expected to show high allele specificity, since the other types 15. Herskowitz, I. 1987. Functional inactivation of genes by domi- of suppressors, although they may actually affect the inter- nant negative mutations. Nature (London) 329:219-222. action with the switch, act more generally on all switches 16. Hess, J. F., K. Oosawa, N. Kaplan, and M. I. Simon. 1988. and do not specifically restore a disrupted interaction. Phosphorylation of three proteins in the signaling pathway of Absolute demonstration of interaction bacterial chemotaxis. Cell 53:79-87. physical between 17. Hess, J. F., K. Oosawa, P. Matsumura, and M. I. Simon. 1987. CheY and the flagellar switch components requires direct Protein phosphorylation is involved in bacterial chemotaxis. biochemical approaches. While it may eventually be possible Proc. Natl. Acad. Sci. USA 84:7609-7613. to determine which proteins interact and where their inter- 18. Huffaker, T. C., M. A. Hoyt, and D. Botstein. 1987. Genetic action sites are, such experiments may prove difficult be- analysis of the yeast cytoskeleton. Annu. Rev. Genet. 21:259- cause of the complexity of the switch apparatus. In addition, 284. genetic approaches to the problem will be greatly enhanced 19. Jarvik, J., and D. Botstein. 1975. Conditional-lethal mutations by a more direct assay for chemotaxis. This information will that suppress genetic defects in morphogenesis by altering resolve the nature of CheY activity at the switch and is structural proteins. Proc. Natl. Acad. Sci. USA 72:2738-2742. crucial to elucidation of the true molecular mechanism that 20. Koop, A. H., M. E. Hartley, and S. Bourgeois. 1987. A low-copy- number vector utilizing 3-galactosidase for the analysis of gene underlies chemotactic behavior. control elements. Gene 52:245-256. 21. Kossmann, M., C. Wolff, and M. D. Manson. 1988. Maltose ACKNOWLEDGMENTS chemoreceptor of Eschenichia coli: interaction of maltose-bind- We thank the following people for contributions to this work: ing protein and the Tar signal transducer. J. Bacteriol. 170:4516- J. Beman for cloning cheY1610 and cheY2885 into mini-Mu, D. 4521. VOL. 174, 1992 CheY SIGNALING SURFACE 6255

22. Kuo, S. C., and D. E. Koshland, Jr. 1989. Multiple kinetic states 41. Sanders, D. A., B. L. Gillece-Castro, A. M. Stock, A. L. for the flagellar motor switch. J. Bacteriol. 171:6279-6287. Burlingame, and D. E. Koshland, Jr. 1989. Identification of the 23. Liu, J., and J. S. Parkinson. 1991. Genetic evidence for inter- site of phosphorylation of the chemotaxis response regulator action between the CheW and Tsr proteins during chemorecep- protein, CheY. J. Biol. Chem. 264:21770-21778. tor signaling by Escherichia coli. J. Bacteriol. 173:4941-4951. 42. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- 24. Lukat, G. S., B. H. Lee, J. M. Mottonen, A. M. Stock, and J. B. ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. Stock. 1991. Roles of the highly conserved aspartate and lysine USA 74:5463-5467. residues in the response regulator of bacterial chemotaxis. J. 43. Slauch, J. M., F. D. Russo, and T. J. Silhavy. 1991. Suppressor Biol. Chem. 266:8348-8354. mutations in rpoA suggest that OmpR controls transcription by 25. Magariyama, Y., S. Yamaguchi, and S.-I. Aizawa. 1990. Genetic direct interaction with the a subunit of RNA polymerase. J. and behavioral analysis of flagellar switch mutants of Salmo- Bacteriol. 173:7501-7510. nella typhimurium. J. Bacteriol. 172:4359-4369. 44. Sockett, H., S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. 26. Martin, C., S. Okamura, and R. Young. 1990. Genetic explora- Macnab. 1992. Molecular analysis of the flagellar switch protein tion of interactive domains in RNA polymerase II subunits. FliM of Salmonella typhimurium. J. Bacteriol. 174:793-806. Mol. Cell. Biol. 10:1908-1914. 45. Spurlino, J. C., G.-Y. Lu, and F. A. Quiocho. 1991. The 2.3-A 27. Matsumura, P., J. J. Rydel, R. Linzmeier, and D. Vacante. 1984. resolution structure of the maltose- or maltodextrin-binding Overexpression and sequence of the Escherichia coli cheYgene protein, a primary receptor of bacterial active transport and and biochemical activities of the CheY protein. J. Bacteriol. chemotaxis. J. Biol. Chem. 266:5202-5219. 160:36-41. 46. Stewart, R. C., and F. W. Dahlquist. 1987. Molecular compo- 28. McNally, D. F., and P. Matsumura. 1991. Bacterial chemotaxis nents of bacterial chemotaxis. Chem. Rev. 87:997-1025. signaling complexes: formation of a CheA/CheW complex en- 47. Stock, A. M., J. M. Mottonen, J. B. Stock, and C. E. Schutt. hances autophosphorylation and affinity for CheY. Proc. Natl. 1989. Three-dimensional structure of CheY, the response regu- Acad. Sci. USA 88:6269-6273. lator of bacterial chemotaxis. Nature (London) 337:745-749. 29. Morris, N. R., M. H. Lai, and C. E. Oakley. 1979. Identification 48. Stock, J. B., A. M. Stock, and J. M. Mottonen. 1990. Signal of a gene for a-tubulin inAspergillus nidulans. Cell 16:437-442. transduction in bacteria. Nature (London) 344:395-400. 30. Mortin, M. 1990. Use of second-site suppressor mutations in 49. Tomizawa, J. 1971. Functional cooperation of genes 0 and P, p. Drosophila to identify components of the transcriptional ma- 549-552. In A. D. Hershey (ed.), The bacteriophage lambda. chinery. Proc. Natl. Acad. Sci. USA 87:4864-4868. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 31. Nakashima, K., K. Kanamaru, A. Hiroflsmi, and T. Mizuno. N.Y. 1991. Signal transduction and osmoregulation in Eschenichia 50. Treptow, N. A., and H. A. Shuman. 1988. Allele-specific malE coli. J. Biol. Chem. 266:10775-10780. mutations that restore interactions between maltose-binding 32. Novick, P., B. C. Osmond, and D. Botstein. 1989. Suppressors of protein and the inner-membrane components of the maltose yeast actin mutations. Genetics 121:659-674. transport system. J. Mol. Biol. 202:809-822. 33. Oosawa, K., J. F. Hess, and M. I. Simon. 1988. Mutants 50a.Vacante, D. Unpublished data. defective in bacterial chemotaxis show modified protein phos- 51. Volz, K., and P. Matsumura. 1991. Crystal structure of Esche- phorylation. Cell 53:89-96. richia coli CheY refined at 1.7 A resolution. J. Biol. Chem. 34. Parkinson, J. S. 1976. cheA, cheB, and cheC genes of Esche- 266:15511-15519. richia coli and their role in chemotaxis. J. Bacteriol. 126:758- 52. Wolfe, A. J., and H. C. Berg. 1989. Migration of bacteria in 770. semisolid agar. Proc. Natl. Acad. Sci. USA 86:6973-6977. 35. Parkinson, J. S. 1978. Complementation analysis and deletion 53. Wolfe, A. J., M. P. Conley, T. J. Kramer, and H. C. Berg. 1987. mapping ofEscherichia coli mutants defective in chemotaxis. J. Reconstitution of signaling in bacterial chemotaxis. J. Bacteriol. Bacteriol. 135:45-53. 169:1878-1885. 36. Parkinson, J. S., and S. E. Houts. 1982. Isolation and behavior 54. Wylie, D., A. Stock, C. Y. Wong, and J. Stock. 1988. Sensory of Eschenichia coli deletion mutants lacking chemotaxis func- transduction in bacterial chemotaxis involves phosphotransfer tions. J. Bacteriol. 151:106-113. between Che proteins. Biochem. Biophys. Res. Commun. 151: 37. Parkinson, J. S., and S. R. Parker. 1979. Interaction of the cheC 891-896. and cheZ gene products is required for chemotactic behavior in 55. Yamaguchi, S., S.-I. Aizawa, M. Kihara, M. Isomura, C. J. Escherchia coli. Proc. Natl. Acad. Sci. USA 76:2390-2394. Jones, and R. M. Macnab. 1986. Genetic evidence for a switch- 38. Parkinson, J. S., S. R. Parker, P. B. Talbot, and S. E. Houts. ing and energy-transducing complex in the flagellar motor of 1983. Interactions between chemotaxis genes and flagellar genes Salmonella typhimunium. J. Bacteriol. 168:1172-1179. in Escherichia coli. J. Bacteriol. 155:265-274. 56. Yamaguchi, S., H. Fujita, A. Ishihara, S.-I. Aizawa, and R. M. 39. Ravid, S., P. Matsumura, and M. Elsenbach. 1986. Restoration Macnab. 1986. Subdivision of flagellar genes of Salmonella of flagellar clockwise rotation in bacterial envelopes by insertion typhimunium into regions responsible for assembly, rotation, of the chemotaxis protein CheY. Proc. Natl. Acad. Sci. USA and switching. J. Bacteriol. 166:187-193. 83:7157-7161. 57. Zhang, Y., C. Conway, M. Rosato, Y. Suh, and M. D. Manson. 40. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Maltose chemotaxis involves residues on the same face of the cloning: a laboratory manual, 2nd ed. Cold Spring Harbor N-terminal and C-terminal domains of maltose-binding protein. Laboratory Press, Cold Spring Harbor, N.Y. J. Biol. Chem., in press.